Real-world advantage and challenge of post-autologous stem cell transplantation MRD negativity in high-risk patients with double-hit multiple myeloma

Background Autologous stem-cell transplantation (ASCT) remains a beneficial approach for patients with newly diagnosed multiple myeloma (NDMM) in the age of novel therapeutic agents. Nevertheless, limited real-world data is available to establish criteria for identifying high-risk ASCT patients. Methods We analyzed outcomes for 168 NDMM patients who underwent ASCT at our center from December 2015 to December 2022. We investigated the impact of the number of high-risk cytogenetics (HRCA), defined as t(4;14), t(14;16), 1q21 gain/amplification, and del(17p), as well as the post-ASCT minimal residual disease (MRD) status as prognostic indicators. We assessed progression-free survival (PFS) and overall survival (OS), and focused on identifying risk factors. Results The cohort included 42% of patients (n = 71) with 0 HRCA, 42% (n = 71) with 1 HRCA, and 16% (n = 26) with ≥ 2 HRCA. After a median follow-up of 31 months, the median PFS was 53 months (95% CI, 37–69), and OS was not reached for the entire cohort. Despite similar rates of MRD-negativity post-ASCT, patients with ≥ 2 HRCA, termed “double hit” (DH), had a significantly higher risk of progression/mortality than those with 0 or 1 HRCA. Multivariate analysis highlighted DH (HR 4.103, 95% CI, 2.046–8.231) and MRD positivity post-ASCT (HR 6.557, 95% CI, 3.217–13.366) as adverse prognostic factors for PFS, with DH also linked to inferior OS. As anticipated, DH patients with post-ASCT MRD positivity displayed the poorest prognosis, with a median PFS of 7 months post-ASCT. Meanwhile, DH patients with MRD negativity post-ASCT showed improved prognosis, akin to MRD-negative non-DH patients. It is noteworthy to exercise caution, as DH patients who initially achieved MRD negativity experienced a 41% cumulative loss of that status within one year. Conclusions This study strongly advocates integrating DH genetic assessments for eligible ASCT patients and emphasizes the importance of ongoing MRD monitoring, as well as considering MRD-based treatment adaptation for those patients in real-world settings. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-024-12077-0.


Introduction
Over the past two decades, there have been significant therapeutic advancements in treating newly diagnosed multiple myeloma (NDMM) [1,2].Despite the introduction of proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies, as well as the use of high-dose chemotherapy and autologous stem-cell transplantation (ASCT), high-risk NDMM patients still face a poor prognosis [3][4][5].With an anticipated increase in options for NDMM, identifying high-risk ASCT patients could inform pre-transplant induction and post-transplant management strategies.The prognosis of MM patients significantly depends on high-risk cytogenetic abnormalities (HRCA) [1,2].Clinical trial data consistently indicate adverse outcomes, particularly for patients with concurrent ≥ 2 genetic lesions deemed highrisk, including t(4;14), t(14;16), 1q21 gain/amplification (1q21+), and del(17p), known as "double hit" (DH) [6][7][8].The presence of minimal residual disease (MRD) following initial therapy is emerging as a powerful prognostic factor in NDMM [9][10][11].However, the significance of combining HRCA number with MRD status as prognostic indicators in real-world ASCT patient population is seldom reported.Our study aims to systematically review the prognostic significance of DH genetics in NDMM patients undergoing standard ASCT at our institution, and to examine the role of post-ASCT MRD status in real-world prognoses.

Patients
We enrolled transplant-eligible patients with NDMM aged 18-65 years with symptomatic, measurable disease defined according to International Myeloma Working Group (IMWG) criteria [12].We retrospectively reviewed electronic records of 205 patients with NDMM who received ASCT at Ruijin Hospital between December 2015 and December 2022.Cut-off for record review was June 4th, 2023.Cytogenetic risk was centrally assessed by fluorescence in-situ hybridization analysis on CD138-positive sorted cells (200 nuclei analyzed) from bone marrow samples at diagnosis.High risk cytogenetic risk (HRCA) included t(4;14), t (14;16), del(17p), 1q21+ (≥ 3 copies) [13].The cutoffs for HRCA positivity were 10% for translocations and 20% for copy number aberrations [14].168 patients with complete baseline cytogenetic data were included in this analysis and were stratified into three subgroups according to the presence of 0, 1, or ≥ 2 HRCA (double hit).
Progression-free survival (PFS) was defined as time from the initialization of therapy, or from stem cell reinfusion (post-ASCT) to progression as per IMWG criteria [12] or death by any cause.Overall survival (OS) was defined as time from the initialization of therapy, or from stem cell re-infusion (post-ASCT) to death by any cause.Patients who did not progress, died, were lost to followup were censored at the time of the last disease assessment.In this retrospective analysis, we investigated the effect of HRCA numbers (0, 1, or ≥ 2) on PFS and OS in the overall patient population.We also assessed the PFS and OS from the date of stem cell re-infusion in patients stratified according to the presence of DH (≥ 2 HRCA) at diagnosis and MRD status after ASCT.Depth of response and relapse criteria were defined as per the IMWG criteria [15].
The limit of detection (LOD) achieved by flow cytometric approach was calculated in each sample according to the following formula: (20/number of viable nucleated cells) × 100 [16].At least one million cellular events for each sample were acquired to reach a theoretical LOD of 2.0 × 10 − 5 .Data were analyzed using the Kaluza Analysis Software to determine the percentage of phenotypically aberrant clonal plasma cells (PCs) in total nucleated cells.MRD positivity was defined as the percentage of phenotypically aberrant clonal PCs equal to or greater than the LOD, otherwise, MRD negativity was considered.MRD negativity refer to CR-based undetectable MRD as per IMWG criteria [15].Evaluation of MRD was done after 4 cycles of induction, before ASCT, within 100 days post-ASCT, and thereafter during follow-up in all patients if possible.For patients reaching MRD negative post ASCT, we monitored MRD status in the bone marrow every 6 months for the first year and yearly thereafter.

Statistics
Time-to-event end points were estimated using the Kaplan-Meier method and compared using pairwise logrank test.Cox proportional hazard regression was used to estimate univariate and multivariate hazard ratios (HR) and 95% confidence intervals (CI).Multivariable logistic regression was used to assess factors associated with post-ASCT MRD status.To assess MRD resurgence or progression across different risk strata, we employed the cumulative incidence function, accounting for death without progression as a competing event.The risks of MRD resurgence or progression were compared using the Gray test.Point estimates for binary endpoints are reported with 95% CI using the Clopper-Pearson exact method, which is based on a binomial distribution.All statistical analyses were conducted using the SPSS software (version 22.0) and R packages survival & survminer in R/Bioconductor (version 3.6.1),with significance defined as p < 0.05.

Clinical and genetic characteristics
From December 2015 to December 2022, our center administered ASCT to 205 patients with NDMM.Among these, 168 patients (82%) with complete cytogenetic data were included in the analysis.1).Enhanced response depths were observed sequentially after induction, following ASCT, during consolidation, and/or throughout the maintenance stage for the respective high-risk groups (Fig. S1).

Genetic contributors to outcome of patients receiving ASCT
To assess the contribution of individual HRCA or their number to the risk status, we plotted these factors against markers of risk such as ISS and time to relapse.The distribution of specific HRCA or their number demonstrated no dependency on ISS stage (Fig. 1A).This suggests a lack of predictability of ISS stage based on specific HRCA or their quantity, and vice versa.The distribution of each HRCA and their number in relation to time to relapse, along with the percentage breakdown of relapse timing, is depicted.Notably, 31% of patients with ≥ 2 HRCA experienced progression within 18 months, significantly higher than those with 0 HRCA (5.6%, p = 0.001) or 1 HRCA (11.2%, p = 0.022) (Fig. 1B).The disparities in early relapse (≤ 18 months) rates based on the number of HRCA suggest a rationale for incorporating DH cytogenetics into predictive models for unfavorable patient outcomes.

Discussions
Our definition of HRCA encompasses 1q21 + along with t(4;14), t(14;16) and del(17p), in line with established standards in the field [7,17,18].In our cohort, 16% patients exhibited DH genetics (≥ 2 HRCA), consistent with the 20% reported in the Master trial [8].Similar to a recent report from MD Anderson, the most common combinations of HRCA in DH cases were [1q+, t(4;14)] and [1q+, del(17p)] [19].Our findings reveal that DH patients had a higher risk of progression or death than patients with 0 or 1 HRCA despite achieving similar rates of MRD-negativity post-ASCT, thereby representing an unmet medical need.The increased risk in DH patients could be attributed to both worse outcomes in those who remain post-ASCT MRD-positive and a higher rate of MRD negativity loss, even in those who initially reached post-ASCT MRD negativity.Importantly, in multivariate analysis, the presence of ≥ 2 HRCA has emerged as an independent prognostic factor, thereby establishing a robust association between DH genetics and poor outcomes in the context of ASCT.We also observed that a bortezomib-based regimen combined with ASCT as standard treatment for NDMM resulted in comparable PFS and OS rates in patients with 0 or 1 HRCA, effectively neutralizing the elevated risk of progression or death associated with a single HRCA as a whole.Although our data suggest that 1q21 + predicts inferior PFS and that t(4;14) is associated with worse OS in univariate analysis, it is noteworthy that 30% of 1q21 + cases and 80% of t(4;14) cases fell within the DH category.[20][21][22], the prognostic significance of a singular occurrence in the context of ASCT is still undefined.Our analysis did not reveal a negative impact of single 1q21 + on prognosis compared to those with 0 HRCA among ASCT patients.Given the small number of patients (n = 5), the detrimental effect of single t(4;14) compared to 0 HRCA on OS requires further investigation.Recent results from the FORTE trial, which involved carfilzomib-based triplet regimens with ASCT as initial treatment, validated a worse PFS in patients with ≥ 2 HRCA [7].
Our analysis shows that stratification by the number of HRCA (DH vs. non-DH) and MRD status (MRDnegative vs. MRD-positive) enhances risk classification further.This stratification uncovers a subgroup with excellent and comparable PFS and OS outcomes, notably those who attain MRD negativity post-ASCT, irrespective of DH status, and another with an absolutely inferior prognosis -DH-positive and post-ASCT MRD-positive (DH + MRD+) patients.This observation indicates that achieving MRD negativity after ASCT could alleviate or even neutralize DH risk factors established at diagnosis.Supporting this notion, a recent meta-analysis covering studies from January 1990 to January 2016 demonstrated that MRD negativity was found to confer an approximate 50% relative reduction in both progression and mortality risk [23].These findings, along with our analysis showing that disease stage and cytogenetic risk profiles had no significant impact on post-ASCT MRD negativity rates (Fig. S6), underscore the necessity of discussing prognosis with patients not only at diagnosis but also throughout treatment phases, as their prognosis could be substantially altered by MRD negative status.Given the dismal prognosis for MRD-positive DH patients, enhancing induction regimens is essential.In quadruplet regimens combined with ASCT, exemplified by CASSIOPEIA [24] and GRIFFIN [25] trials, the incorporation of CD38 Ab into induction protocols greatly increased MRD negativity post-ASCT, from 44% with VTd to 64% with D-VTd, or from 20% with VRd to 51% with D-VRd.Furthermore, the MASTER study revealed that 79% of DH patients achieved MRD negativity by NGS (cutoff 10 − 5 ) following Dara-KRd induction, ASCT, and Dara-KRd consolidation [8].Considering that some DH patients remain MRDpositive despite intensified induction and ASCT, pioneering treatments like chimeric antigen receptor T-cells (CART) and bispecific T-cell engagers moving to frontline, may be justified.As shown in our earlier reports, B-cell maturation antigen-targeting (BCMA)-CART is promising, offering deep remission (72.9% MRD-negative CR, cutoff 10 − 5 ) and a favorable long-term safety profile in relapsed/refractory MM [26][27][28].
Nevertheless, achieving MRD negativity post-ASCT in DH patients has been shown to be insufficient.Our findings suggest that it is challenging for DH patients to sustain MRD negativity.One potential explanation for this may be the limited sensitivity of flow cytometry sensitivity (2 × 10 − 5 ) employed in our study.However, the MAS-TER trial, implementing a higher sensitivity threshold of < 10 − 5 by NGS, also reported a higher risk of losing MRD negativity in MRD-negative DH patients.Similarly, the FORTE trial [7], used the same MRD sensitivity as MAS-TER, revealing a significantly lower rate of 1-year sustained MRD negativity in DH patients.Whereas, those DH who maintained 1-year sustained MRD negativity showed a 4-year PFS comparable to patients with 0 or 1 HRCA [7].This underscores the critical importance of sustained MRD negativity as a treatment goal for DH patients.With a longer median follow-up in our study, it is plausible that DH + MRD-patients might exhibit a significantly distinct survival curve from those DH-MRD-, owing to a lower rate of sustained MRD negativity.As a result, we strongly advise against treatment deintensification for DH patients solely based on achieving MRD negativity, particularly outside of clinical trial settings.Furthermore, post-transplant changes in MRD status across varying numbers of HRCA highlight the necessity for serial MRD assessments in initially MRD-negative patients to better assess progression risk.While the introduction of CD38 Ab-based quadruplet regimens is likely to enhance the initial response rates in DH patients, maintaining sustained MRD negativity requires tailored post-ASCT intensified treatment.Analyses from the MASTER, FORTE, and UK OPTIMUM/MUKnine trials advocate risk-adapted post-ASCT consolidation for this patient cohort [8,29,30].Moreover, adding carfilzomib to lenalidomide maintenance extends PFS beyond lenalidomide alone, across all cytogenetic risk groups in the FORTE [7], further supporting intensified maintenance for DH patients.This includes using a doublet of a proteasome inhibitor and an immunomodulatory drug or even more potent combinations.
A limitation of our study is that patients received PAD induction therapies, with the addition of lenalidomide and/or CD38 Ab later.The initial choice of PAD rather than VRD as the standard of care was pragmatic, based on insurance coverage.Despite different induction regimens, our analysis showed similar and high rates of post-ASCT MRD negativity across various risk strata.Furthermore, the ISKIA trial presented at this year's ASH indicates that the addition of isatuximab to the KRd regimen increased the rate of post-consolidation MRD negativity at the 10 − 5 level to a small extent (77% for IsaKRd vs. 67% for KRd; p = 0.049), but demonstrated a more pronounced difference at the 10 − 6 threshold (67% vs. 48%; p < 0.001), better distinguishing between the two treatment groups.This improvement was consistent among DH patients (10 − 5 : 77% vs. 53%; 10 − 6 : 77% vs. 27%).These findings suggest that deeper MRD thresholds are crucial to accurately identify MRD negativity in patients including in those with DH [31].
In summary, our findings strongly advocate the incorporation of DH genetics assessment for all eligible ASCT patients in real-world scenarios.The data suggest DH patients might initially benefit from achieving post-ASCT MRD negativity via tailored pre-transplant induction, followed by maintaining this negativity through post-transplant consolidation and intensive maintenance.For DH patients, even after initial post-ASCT MRD negativity is achieved, continuous MRD monitoring remains essential.

Fig. 1
Fig. 1 The association of individual HRCA or the number of HRCA with clinical risk groups.(A) The distribution of individual HRCA or the number of HRCA by ISS sorted by the proportion of patients; (B) The contribution of individual HRCA or the number of HRCA to relapse sorted by the proportion of patients, with a breakdown of PFS over ≤ 18 months/ 18-36 months/>36 months, or no progression.HR, high risk; HRCA, high-risk cytogenetics; PD, progressive disease

Fig. 2
Fig. 2 Kaplan-Meier plots for PFS and OS for NDMM patients treated with standard of care ASCT.(A, B) PFS (A) and OS (B) for the entire cohort of 168 patients; (C, D) PFS (C) and OS (D) stratified by the number of HRCA (0 vs. 1 vs. ≥2 HRCA).HRCA, high-risk cytogenetics; PFS, progression free survival; OS, overall survival

Fig. 3
Fig. 3 Univariate and multivariate analyses.(A, B) Associations of clinical factors and the number of HRCA with PFS (A) and OS (B) in univariate analyses; (C, D) Associations of clinical factors and the number of HRCA with PFS (C) and OS (D) in multivariate analyses.HRCA, high-risk cytogenetics; HR, hazard ratio; CI, confidence interval; PFS, progression free survival; OS, overall survival; HR was transformed into log 10 (HR) in the Figure

Fig. 4
Fig. 4 Response to therapy, by number of cycles and follow-up in the subset of 26 DH (≥ 2HRCA) patients.P, patient; C, cycle; m, months; y, years; PR, partial remission; VGPR, very good partial remission; CR, complete remission; PD, progressive disease

Fig. 5
Fig. 5 Prognosis of patients according to the presence of DH HRCA and post-ASCT MRD status.(A, B) Probability of PFS (A) and OS (B) adjusted for the presence of DH HRCA and post-ASCT MRD status; (C) Cumulative incidence of progression or MRD resurgence for patients stratified by the number of HRCA after achieving post-ASCT MRD negativity.DH, double hit; MRD, minimal residual disease; HRCA, high-risk cytogenetics; PFS, progression free survival; OS, overall survival